Francesca Cappitelli

Biotechnology applied to cultural heritage: biorestoration of frescoes using viable bacterial cells and enzymes

Aims:  To set up and employ, for the biorestoration of cultural heritage (altered frescoes), an advanced and innovative biotechnology method based on the sequential use of whole viable bacterial cells and specific enzymes.

Advantages of Using Microbial Technology over Traditional Chemical Technology in Removal of Black Crusts from Stone Surfaces of Historical Monuments

ABSTRACT This study compares two cleaning methods, one involving an ammonium carbonate-EDTA mixture and the other involving the sulfate-reducing bacterium Desulfovibrio vulgaris subsp. vulgaris ATCC 29579, for the removal of black crust (containing gypsum) on marble of the Milan Cathedral (Italy). In contrast to the chemical cleaning method, the biological procedure resulted in more homogeneous removal of the surface deposits and preserved the patina noble under the black crust. Whereas both of the treatments converted gypsum to calcite, allowing consolidation, the chemical treatment also formed undesirable sodium sulfate.

Improved Methodology for Bioremoval of Black Crusts on Historical Stone Artworks by Use of Sulfate-Reducing Bacteria

ABSTRACT An improved methodology to remove black crusts from stone by using Desulfovibrio vulgaris subsp. vulgaris ATCC 29579, a sulfate-reducing bacterium, is presented. The strain removed 98% of the sulfates of the crust in a 45-h treatment. Precipitation of black iron sulfide was avoided using filtration of a medium devoid of iron. Among three cell carriers, Carbogel proved to be superior to both sepiolite and Hydrobiogel-97, as it allowed an easy application of the bacteria, kept the system in a state where microbial activity was maintained, and allowed easy removal of the cells after the treatment.

Microorganisms Attack Synthetic Polymers in Items Representing Our Cultural Heritage

With advancements in materials science over the past few decades, there has been a dramatic increase in the use of synthetic polymers by both artists and conservators. Synthetic polymers in items representing our cultural heritage occur either as original constituents of works of art or as materials used for conservation treatment, and these polymers include adhesives, consolidants, and protective coatings. In the 1980s there was a change in the perception of plastics from consumer goods and disposable materials to fashionable, highly collectable pieces with historical and technological significance (27, 34). Now, in their 20th and 21st century collections, most museums and galleries possess objects made from the thousands of different plastics that have been produced. As museums keep acquiring objects that reflect both everyday life and technological and historical events, the proportion of plastics in museums is increasing dramatically. Plastics may be present in objects of everyday life, such as housewares, jewelry, equipment, furniture, information technology, photography, and toys, and more of these objects are entering museum collections and contemporary art (57). In addition, synthetic polymers have been widely employed for treatment of items representing our cultural heritage as adhesives, consolidants, and protective coatings to preserve many artifacts from further deterioration (20, 45). Synthetic polymer conservation has been formally recognized as a research area only since the 1990s, and it was in this period that the worlds most important organization in the field of cultural heritage conservation, the Committee for Conservation of the International Council of Museums, established the Modern Materials and Contemporary Art Working Group. Indeed, owners and curators have begun to notice that objects made of plastics degrade with time, sometimes very rapidly. Importantly, many synthetic polymers appear to deteriorate faster than other materials in museum collections and have a useful lifetime of just decades (57). Synthetic polymeric materials can suffer different forms of deterioration, including chemical (e.g., oxidation), physical (e.g., UV light), and biological. Although many reports in the scientific literature claim that microorganisms are capable of degrading synthetic resins (35, 42, 59, 68), the microbial contamination of synthetic polymers that are used as materials for conservation treatment (29, 32) and in contemporary collections (50) is still underestimated. Indeed, it was only in the 2005-2008 program that the Committee for Conservation of the International Council of Museums Modern Materials and Contemporary Art Working Group embraced “(microbial) biodeterioration” as a research topic (http://icom-cc.icom.museum/Documents/WorkingGroup/ModernMaterials/Modern-materials2005-2008.pdf). Microorganisms can damage the structure and function of synthetic polymers. According to Flemming (22), the main types of damage include (i) biological coating masking surface properties, (ii) increased leaching of additives and monomers that are used as nutrients, (iii) production of metabolites (e.g., acids), (iv) enzymatic attack, (v) physical penetration and disruption, (vi) water accumulation, and (vii) excretion of pigments. Table ​Table11 describes microorganisms and their modes of action for degrading synthetic resins (polyvinyl chloride [PVC], polyurethane, nylon, and acrylics). Barbie dolls, together with many other toys, clothes, and electrical insulation found in museums, are made from PVC (58). The instability of plasticized PVC is frequently manifested as migration of the plasticizers. Colonization of PVCs by fungi, especially black fungi, due to the availability of platicizers on the surface has been assessed several times (30, 51, 67). Webb et al. (67) identified fungal isolates obtained from PVC by PCR amplification and partial sequencing of the internally transcribed spacer regions and the 5.8S rRNA gene or the V3 domain of the 28S rRNA gene. TABLE 1. Microorganisms degrading the synthetic polymers PVC, polyurethane, nylon, and acrylics and their mode of action It has been suggested that biodeterioration of polyurethane polymers, which are products of a polyol based on either a polyester or polyether and a di- or polyisocyanate, occurs through enzymatic action of hydrolases, such as ureases, proteases, and esterases (18, 21, 52). Degradation of polyurethanes by microorganisms in 20th century museum textiles has been reported by many researchers (36, 63). Polyurethanes can also be found in products such as furniture, adhesives, paints, elastomers, coatings, and contemporary art (33, 34, 52). Biodeterioration due to enzymes, presumably including a manganese peroxidase of the basidiomycete Bjerkandera adusta, was also observed for the aliphatic polyamide Nylon-6 fiber (24). Damage to the polymer was assessed by microscopic examination, differential scanning calorimetry, and evaluation of changes in viscosity. One of the reasons for introducing synthetic consolidants and protective compounds in conservation treatments was the expectation that these materials would be more resistant to microbial attack than natural organic products. In 1968 the superintendents at Ostia Antica (Rome, Italy) decided to replace natural organic compounds, which are easily degraded by microorganisms, with acrylic compounds in conservation treatments. Frescoes detached with Paraloid did not show any biodeterioration problem for the first 3 years after application (4). However, as early as the 1950s, some experiments on biodeterioration of polyvinyl acetate resins were reported by the Istituto Centrale del Restauro in Rome, Italy (26). Generally, filamentous fungi were the agents causing deterioration of these materials that were studied the most, especially in early experiments (17, 41, 54, 61), although some bacteria, yeasts, algae, and lichens that are capable of growing on synthetic polymers have been found or isolated (14). Historically, identification of filamentous fungal species has been based on morphological characteristics, both macroscopic and microscopic. These methods may often be time-consuming and inaccurate, which has required the development of identification protocols that are rapid, sensitive, and precise. In the last decade molecular approaches for rapid characterization of fungi on painted items representing our cultural heritage and paint coatings have been developed (46, 53). A protocol for efficient extraction of fungal DNA from micromycetes colonizing painted art objects was developed by Mohlenhoff et al. (46), who claimed to have successfully removed any inhibitors. In particular, melanin can also be present, which is highly resistant to UV light, enzymatic digestion, and chemical breakdown and might be a potent inhibitor of DNA amplification (46). PCR amplification of the 28S rRNA gene and denaturing gradient gel electrophoresis analysis were used to characterize fungal communities. According to Saad et al. (53), fungi are commonly found on paint films as spores other than mycelium; hence, it is necessary to ensure that DNA extraction is effective also for propagules. The method used involves spore lysis by incubation of a specimen with the enzyme Lyticase, followed by bead beating. DNA is then purified from the lysate with a QIAamp DNA mini kit (53). There have also been case studies related to biodeterioration agents other than filamentous fungi. Bacterial biofilms composed of Pseudomonas aeruginosa, Ochrobactrum anthropi, Alcaligenes denitrificans, Xanthomonas maltophila, and Vibrio harveyi formed readily on the surfaces of synthetic materials being considered for use in space applications (28). A yeast isolated from a bronze statue treated with the acrylic-based coating Incralac was found to accelerate the deterioration of the coating itself, as determined by scanning electron microscopy and electrochemical impedance spectroscopy (44). Stones impregnated with Ahydrosil Z, a silicone resin, were recolonized by algae and fungi more quickly than untreated specimens (40). Rapid recolonization by the alga Stichococcus bacillaris was also noticed in the Roman archaeological site at Luni in northern Italy after treatment with an epoxy resin and an acrylic-siliconic resin (19). Finally, lichens were reported to deteriorate a synthetic polyester resin that was used as a consolidant of stucco walls and column capitals in the Roman city Baelo Claudia in Spain (2). The ecological succession of fungi over 10 months on two Brazilian buildings painted with a white acrylic paint was described by Shirakawa et al. (60). Prior to painting, the walls were treated with hypochlorite. In addition to Cladosporium, the main fungal genus identified during the experiment, the other fungal genera detected were Alternaria, Curvularia, Epicoccum, Helminthosporium, Coelomycetes, Monascus, Nigrospora, and Aureobasidium. The yeast population fell to undetectable levels after the third week, and this microbial group was not detected again until 7 months, after which the number of cells increased.

From Papyrus to Compact Disc: The Microbial Deterioration of Documentary Heritage

Abstract Highly significant evidence of the intellectual and cultural efforts of the human race is contained in documents. They take many forms, from papyri through paper to modern magnetic media and optical records. These items are mainly made of organic materials many of which contain polymers, which span from cellulose and its derivatives to synthetic resins. As with other manmade objects, however, documentary heritage is susceptible to chemical, physical, and biological damage. For the colonization and establishment of any biological community, the composition of materials used, their status of conservation, and environmental and climatic factors, such as temperature and humidity, are important elements to take into account. This article covers the scientific investigation of microbial degradation of documents, which is one of the most serious and underappreciated sources of damage to library and archival materials. In particular, although less known, modern records, including compact discs, are also subjected to biodeterioration. Archival and library material preservation broadly encompasses those activities and functions designed to produce a suitable and safe environment that extends the life of collections in useable condition for as long as is feasible. In the literature quoted, key information is also provided to avoid or limit microbial growth and some conservation treatments are also reported.

Feasibility of Removing Surface Deposits on Stone Using Biological and Chemical Remediation Methods

The study was conducted on alterations found on stone artwork and integrates microbial control and a biotechnological method for the removal of undesirable chemical substances. The Demetra and Cronos sculptures are two of 12 stone statues decorating the courtyard of the Buonconsiglio Castle in Trento (Italy). An initial inspection of the statues revealed putative black crusts and highlighted the microbial contamination causing discoloration. In 2006, the Cultural Heritage Superintendence of Trento commissioned us to study and remove these chemical and biological stains. Stereomicroscopy characterised the stone of the sculptures as oolitic limestone, and infrared analyses confirmed the presence of black crusts. To remove the black crusts, we applied a remediation treatment of sulphate-reducing bacteria, which removes the chemical alteration but preserves the original stone and the patina noble. Using traditional and biomolecular methods, we studied the putative microbial contamination and confirmed the presence of biodeteriogens and chose biocide Biotin N for the removal of the agents causing the discolouration. Denaturing gradient gel electrophoresis fluorescent in situ hybridisation established that Cyanobacteria and green algae genera were responsible for the green staining whereas the black microbial contamination was due to dematiaceous fungi. After the biocide Biotin N treatment, we applied molecular methods and demonstrated that the Cyanobacteria, and most of the green algae and dematiaceous fungi, had been efficiently removed. The reported case study reveals that conservators can benefit from an integrated biotechnological approach aimed at the biocleaning of chemical alterations and the abatement of biodeteriogens.

Biofilm Formation in Food Processing Environments is Still Poorly Understood and Controlled

The presence of undesirable biofilms on food processing contact surfaces may lead to: (1) transmission of diseases; (2) food spoilage; (3) shortened time between cleaning events; (4) contamination of product by nonstarter bacteria; (5) metal corrosion in pipelines and tanks; (6) reduced heat transfer efficacy or even obstruction of the heat equipment. Despite the significant problems caused by biofilms in the food industry, biofilm formation in these environments is still poorly understood and effective control of biofilms remains challenging. Although it is understood that cell attachment and biofilm formation are influenced by several factors, including type of strain, chemical–physical properties of the surface, temperature, growth media and the presence of other microorganisms, some conflicting statements can be retrieved from the literature and there are no general trends yet that allow us to easily predict biofilm development. It is likely that still unexplored interaction of factors may be more critical than the effect of a single parameter. New alternative biofilm control strategies, such as biocontrol, use of enzymes and phages and cell-to-cell communication interference, are now available that can reduce the use of chemical agents. In addition, as preventing biofilm formation is a more efficient strategy than controlling mature biofilm, the use of surface-modified materials have been suggested. These strategies may better reveal their beneficial potential when the ecological complexity of biofilms in food environments is addressed.

Mineral-microbe interactions: biotechnological potential of bioweathering.

Mineral-microbe interaction has been a key factor shaping the lithosphere of our planet since the Precambrian. Detailed investigation has been mainly focused on the role of bioweathering in biomining processes, leading to the selection of highly efficient microbial inoculants for the recovery of metals. Here we expand this scenario, presenting additional applications of bacteria and fungi in mineral dissolution, a process with novel biotechnological potential that has been poorly investigated. The ability of microorganisms to trigger soil formation and to sustain plant establishment and growth are suggested as invaluable tools to counteract the expansion of arid lands and to increase crop productivity. Furthermore, interesting exploitations of mineral weathering microbes are represented by biorestoration and bioremediation technologies, innovative and competitive solutions characterized by economical and environmental advantages. Overall, in the future the study and application of the metabolic properties of microbial communities capable of weathering can represent a driving force in the expanding sector of environmental biotechnology.

Synthetic Consolidants Attacked by Melanin-Producing Fungi: Case Study of the Biodeterioration of Milan (Italy) Cathedral Marble Treated with Acrylics

ABSTRACT Monuments and artistic stone surfaces are often consolidated and protected with synthetic polymers, in particular, acrylics. Although it is generally thought that acrylic polymers are resistant to biodeterioration, we report for the first time the systematic occurrence of dematiaceous meristematic fungi on many marble samples of the cathedral in Milan (Italy) previously treated with this material. Fourier transform infrared spectroscopy applied to the Milan cathedral stone samples revealed characteristic features of biodeteriorated synthetic resins that differentiated them from the aged but nonbiodeteriorated samples. Samples showing biological colonization were analyzed for the presence of fungi. Cultivation and morphological characterization and methods independent from cultivation, such as denaturing gradient gel electrophoresis coupled with partial 18S rRNA gene sequencing and immunofluorescence staining with melanin-binding antibodies, showed that melanin-producing species are heavily present on stone surfaces protected with acrylic resins. This observation raises the question of the effectiveness of acrylics in protecting stone artworks.

Hindering biofilm formation with zosteric acid.

The antifoulant, zosteric acid, was synthesized using a non-patented process. Zosteric acid at 500 mg l−1 caused a reduction of bacterial (Escherichia coli, Bacillus cereus) and fungal (Aspergillus niger, Penicillium citrinum) coverage by 90% and 57%, respectively. Calculated models allowed its antifouling activity to be predicted at different concentrations. Zosteric acid counteracted the effects of some colonization-promoting factors. Bacterial and fungal wettability was not affected, but the agent increased bacterial motility by 40%. A capillary accumulation test showed that zosteric acid did not act as a chemoeffector for E. coli, but stimulated a chemotactic response. Along with enhanced swimming migration of E. coli in the presence of zosteric acid, staining showed an increased production of flagella. Reverse transcriptase-PCR revealed an increased transcriptional level of the fliC gene and isolation and quantification of flagellar proteins demonstrated a higher flagellin amount. Biofilm experiments confirmed that zosteric acid caused a significant decrease in biomass (−92%) and thickness (−54%).